Proteolytically cleavable fusion proteins with high molar specific activity

ABSTRACT

The invention relates to therapeutic fusion proteins in which a coagulation factor is fused to a half-life enhancing polypeptide, and in which both are connected by a linker peptide that is proteolytically cleavable. The cleavage of such linkers liberates the coagulation factor from activity-compromising steric hindrance caused by the half-life enhancing polypeptide and thereby allows the generation of fusion proteins may show relatively high molar specific activity when tested in coagulation-related assays. Furthermore, the fact that the linker is cleavable can enhance the rates of inactivation and/or elimination after proteolytic cleavage of the peptide linker compared to the rates measured for corresponding therapeutic fusion proteins linked by the non-cleavable linker having the amino acid sequence GGGGGGV.

This application is a continuation-in-part of U.S. application Ser. No.11/812,016, filed Jun. 14, 2007, which claims priority to U.S.Provisional Application No. 60/819,620, filed Jul. 11, 2006. Theapplication also claims priority to European Patent Application No.06012262.9, filed Jun. 14, 2006.

The present invention relates to the field of modified therapeuticfusion proteins with increased half-life compared to their non-modifiedparent therapeutic polypeptides. The invention, for example, relates tocoagulation factors fused to half-life enhancing polypeptides (HLEPs),which are connected by linker peptides that are proteolyticallycleavable. The cleavage of such linkers liberates the therapeuticpolypeptide from activity-compromising steric hindrance caused by theHLEP and thereby allows the generation of fusion proteins, which retaina high molar specific activity of the coagulation factor. When thetherapeutic fusion proteins are zymogens, one may use linkers thatliberate the therapeutic polypeptide essentially simultaneously with itsactivation in vivo upon exposure to the corresponding protease(s).Embodiments of the present invention may show a faster inactivation rateof a given coagulation factor once the coagulation factor is activatedand the peptide linker is proteolytically cleaved in acoagulation-related mode and/or a faster elimination rate of a givencoagulation factor once the coagulation factor is activated and thepeptide linker is proteolytically cleaved in a coagulation-related mode,compared to the corresponding fusion protein without cleavable linker.

One aspect of the invention is demonstrated for example by human vitaminK-dependent polypeptides Factor IX, Factor VII, and Factor VIIa. Thesame concept also may be applied to other coagulation factors. Anyhalf-life enhancing polypeptide (HLEP) may be connected to thetherapeutic polypeptide by a cleavable linker peptide. For instancealbumin or immunoglobulins or fragments derived therefrom without anantigen binding domain, such as the Fc fragment, may serve as HLEPs. Theinvention also relates to cDNA sequences coding for the therapeuticpolypeptides and derivatives thereof genetically fused to a cDNA codingfor HLEPs, such as human serum albumin linked by oligonucleotides thatcode for cleavable, intervening peptide linkers. Such encodedderivatives may exhibit improved half-life and molar specific activitiesthat are increased in comparison to their non-cleavable counterparts.The invention also relates to recombinant expression vectors containingsuch cDNA sequences, host cells transformed with such recombinantexpression vectors, recombinant polypeptides and derivatives which mayhave biological activities comparable to the unmodified wild typetherapeutic polypeptide, but having improved half-lifes. The inventionalso relates to processes for the manufacture of such recombinantproteins and their derivatives. The invention also relates to a transfervector for use in human gene therapy, which comprises such modified DNAsequences, which can be useful to increase half-life in vivo.

Several recombinant, therapeutic polypeptides are commercially availablefor therapeutic and prophylactic use in humans. The patients in generalmay benefit from the specific mode of action of the recombinant activeingredients but a disadvantage may be their limited availability due toexpensive and complex manufacturing processes. A reduction of thenecessary dose or the frequency of administration of such products couldimprove this situation. A reduced frequency of administration couldimprove the convenience for the patient and, therefore, also theacceptance of the therapy. Several ideas have been described to attemptto achieve the goal of an increased in vivo half-life afteradministration. See, e.g. Ballance et al. (WO 01/79271), Sheffield etal. (Sheffield W. P. et al. (2004), Br. J. Haematol. 126: 565-573), WO2002/04598, WO 2003/059935, WO 2004/081053, WO 2004/101740, WO2005/001025, WO 91/09125, and WO 03/068934. Fusions of coagulationfactors to half-life enhancing polypeptides have been suggested tolengthen the half-life of coagulation factors administered to patients.However, once a coagulation factor is activated during coagulationeither by proteolytic cleavage of the zymogen (like FIX) or by contactof an already proteolytically “pre”-activated factor to a secondpolypeptide (like FVIIa binding to Tissue Factor), it may no longer bedesirable to maintain the long half-life of the now activatedcoagulation factor, as this might lead to thrombotic complications. Thisis the case for a wild type coagulation factor such as FVIIa (Aledort L.M., J Thromb Haemost 2(10): 1700-1708 (2004)) and may be even morerelevant if the activated factor has an increased half-life. It istherefore one objective of some embodiments of the present invention toprovide long-lived coagulation factors, which after activation or afteravailability of a cofactor have a half-life comparable to that of anunmodified coagulation factor.

Fusions of the coagulation factors to half-life enhancing polypeptidesas described in the prior art and as also shown in examples 6 and 7 maysuffer in general from a reduced molar specific activity of the fusedcoagulation factor. Another aspect of the present invention is toprovide coagulation factors with enhanced half-life that show increasedmolar specific activity compared to the corresponding therapeutic fusionprotein without a cleavable linker.

Some embodiments of this invention include therapeutic fusion proteinscomprising:

-   -   a) a coagulation factor,    -   b) a half-life enhancing polypeptide (HLEP) chosen from        albuminand immunoglobulin, and    -   c) a peptide linker, which linker joins the coagulation factor        and the half-life enhancing polypeptide;

wherein the peptide linker is cleavable by proteases involved incoagulation or proteases activated by coagulation enzymes, and whereinthe therapeutic fusion protein has, in comparison to the respectivetherapeutic fusion protein linked by a non-cleavable linker having theamino acid sequence GGGGGGV, at least one of the following properties:

-   -   i) an increased molar specific activity in at least one        coagulation-related assay,    -   ii) an increased inactivation rate of the activated coagulation        factor after the peptide linker is proteolytically cleaved in a        coagulation-related mode, and    -   iii) an increased elimination rate of the activated coagulation        factor after the peptide linker is proteolytically cleaved in a        coagulation-related mode.

A “coagulation factor,” as used herein, includes variants or derivativesthereof, such as genetically engineered or chemically modified variantsor active fragments thereof. See below for additional description ofexamples of “coagulation factors.”

“Albumin,” as used herein, includes polypeptides of the albumin familyof proteins such as human serum albumin and bovine serum albumin,including variants and derivatives thereof, such as geneticallyengineered or chemically modified albumin variants and fragments ofalbumin proteins. See below for additional description of examples of“albumin.”

“Immunoglobulin,” as used herein, includes variants and derivatives ofimmunoglobulin proteins, such as genetically engineered or chemicallymodified immunoglobulin variants and fragments of immunoglobulin, forexample, an Fc fragment or other fragment not containing an antigenbinding domain. See below for additional description of examples of“immunoglobulin.”

As a consequence of the cleavable linker, after cleavage of the peptidelinker in a coagulation-related mode the coagulation factor more closelyresembles the behaviour of the native, non-fused factor and does notshow an increased half-life of the active factor with potentiallyprothrombotic effect.

“Proteolytic cleavage in a coagulation-related mode,” as used herein,means any proteolytic cleavage that occurs as a consequence of theactivation of at least one coagulation factor or coagulation cofactor.

The phrase “activated coagulation factor after the peptide linker isproteolytically cleaved in a coagulation-related mode,” as used hereinmeans that the coagulation factor is either activated almost in parallelto the proteolytic cleavage of the linker peptide, or that thecoagulation factor was already activated before the proteolytic cleavageof the linker peptide. Activation may occur, for example by proteolyticcleavage of the coagulation factor or by binding to a cofactor.

A further aspect of the present invention is to provide therapeuticfusion proteins comprising:

-   -   a) a coagulation factor,    -   b) a half-life enhancing polypeptide (HLEP) chosen from albumin        and immunoglobulin, and    -   c) a peptide linker which joins the coagulation factor and the        half-life enhancing polypeptide;

wherein the peptide linker is cleavable by proteases involved incoagulation or activated by coagulation enzymes, and wherein thetherapeutic fusion protein has, in comparison to the respectivetherapeutic fusion protein linked by a non-cleavable linker having theamino acid sequence GGGGGGV, at least one of the following properties:

-   -   i) an increased molar specific activity in at least one        coagulation-related assay,    -   ii) an increased inactivation rate of the activated coagulation        factor after the peptide linker is proteolytically cleaved in a        coagulation-related mode,    -   iii) an increased elimination rate of the activated coagulation        factor after the peptide linker is proteolytically cleaved in a        coagulation-related mode, and    -   iv) an enhanced in vivo recovery as compared to the in vivo        recovery of the unmodified coagulation factor.

Some embodiments include therapeutic fusion proteins which have anenhanced in vivo recovery compared to the unmodified coagulation factorby at least 10%, for example, by at least 25% or by 40% or more.

Exemplary coagulation factors are vitamin-K dependent coagulationfactors, FVIIa, and FIX, and fragments and variants thereof, such asgenetically engineered or chemically modified variants or fragments,such as are described in more detail below.

HLEPs may be albumin and fragments or variants thereof andimmunoglobulins including fragments and variants thereof, as describedabove, and in more detail, below.

The peptide linker in some embodiments may comprise a sequence of thetherapeutic polypeptide to be administered or a variant thereof, whichshould result in a decreased risk of neoantigenic properties (formationof a novel potentially immunogenic epitope due to the occurrence of apeptide within the therapeutic antigen which does not exist in humanproteins) of the expressed fusion protein. Also in case the therapeuticprotein is a zymogen (e.g. needs to be proteolytically activated) thekinetics of the peptide linker cleavage may more closely reflect thecoagulation-related activation kinetics of the zymogen. Thus, in someembodiments, a zymogen and a corresponding peptide linker are activatedand respectively cleaved, with comparable kinetics. For this reason,embodiments of the present invention also relate to fusion proteins of azymogen and a HLEP, where the kinetics of the linker cleavage byrelevant proteases are not delayed by more than a factor of 3, such asnot by more than a factor of 2, compared to the kinetics of the zymogenactivation.

In another embodiment, the peptide linker comprises cleavage sites formore than one protease. This can be achieved, for example, by a peptidelinker that can be cleaved at the same position by different proteasesor by a peptide linker that provides two or more different cleavagesites. This may be advantageous circumstances where the therapeuticfusion protein must be activated by proteolytic cleavage to achieveenzymatic activity and where different proteases may contribute to thisactivation step. This is the case, for example, upon activation of FIX,which can either be achieved by FXIa or by FVIIa/Tissue Factor (TF).

Some embodiments of the invention are therapeutic fusion proteinswherein the peptide linker is cleavable by the protease that normallyactivates the coagulation factor in vivo, thereby ensuring that thecleavage of the linker is linked to the activation of the coagulationfactor at a site at which coagulation occurs.

Other exemplary therapeutic fusion proteins according to the inventionare those wherein the linker is cleavable by the coagulation factorwhich is part of the therapeutic fusion protein once it is activated,thus also ensuring that cleavage of the fusion protein is connected witha coagulatory event.

Other exemplary therapeutic fusion proteins according to the inventionare those wherein the linker is cleavable by a protease, which itself isactivated directly or indirectly by the activity of the coagulationfactor which is part of the therapeutic fusion protein, thus alsoensuring that cleavage of the fusion protein is connected with acoagulatory event.

One class of therapeutic fusion proteins included in this inventioncomprises those wherein the linker is cleavable by FXIa and/or byFVIIa/TF and the coagulation factor is FIX.

For example, embodiments of the invention include fusion proteinscomprising the vitamin K-dependent polypeptide Factor IX, cleavablelinkers, and albumin as the HLEP, as well as their corresponding cDNAsequences. The invention also relates to cDNA sequences coding for anyother coagulation factors which can be proteolytically activated or thatare involved in the activation of other zymogens or polypeptides. Insome embodiments, these cDNAs are genetically fused to cDNA sequencescoding for human serum albumin or other HLEPs, and are linked byoligonucleotides that code for intervening, cleavable peptide linkers.The expressed therapeutic fusion proteins may exhibit molar specificactivities which are increased in comparison to their non-cleavablecounterparts. The invention also relates to recombinant expressionvectors containing such fused cDNA sequences, host cells transformedwith such recombinant expression vectors, recombinant therapeutic fusionproteins and derivatives that may have biological activities almostcomparable to the unmodified wild type therapeutic polypeptides buthaving improved in vivo half-life. The invention also relates toprocesses for the manufacture of the recombinant polypeptides of theinvention and their derivatives. The invention also relates to atransfer vector for use in human gene therapy, which comprises suchmodified DNA sequences useful to increase product levels in vivo.

Some therapeutic fusion proteins according to the invention have a molarspecific activity, in particular a molar specific activity in at leastone coagulation-related assay, that is at least 25% increased comparedto that of the corresponding therapeutic fusion protein without acleavable linker. Other therapeutic fusion proteins have a molarspecific activity that is increased by at least 50%, or by at least100%, in at least one of the different coagulation-related assaysavailable, compared to that of the corresponding therapeutic fusionprotein without a cleavable linker, such as one with the non-cleavablesequence GGGGGGV.

Additional embodiments of the present invention are therapeutic fusionproteins wherein the inactivation rate of the activated coagulationfactor after cleavage of the peptide linker which links the coagulationfactor to the half-life enhancing polypeptide is increased by at least10% as compared to the inactivation rate of the activated coagulationfactor in a corresponding therapeutic fusion protein without a cleavablelinker. Other embodiments are therapeutic fusion proteins in which theinactivation rate is increased by at least 25%, or by at least 50% ascompared to the inactivation rate of the activated coagulation factor ina corresponding therapeutic fusion protein without a cleavable linker,such as one with the non-cleavable sequence GGGGGGV.

Additional embodiments of the present invention are therapeutic fusionproteins wherein the elimination rate of the coagulation factor aftercleavage of the peptide linker that links the coagulation factor to thehalf-life enhancing polypeptide is increased by at least 10% as comparedto the elimination rate of the coagulation factor in a correspondingtherapeutic fusion protein without a cleavable linker. Other embodimentsare therapeutic fusion proteins in which the elimination rate isincreased by at least 25%, or by at least 50% as compared to theelimination rate of the coagulation factor in a correspondingtherapeutic fusion protein without a cleavable linker, such as one withthe non-cleavable sequence GGGGGGV.

Vitamin K-Dependent Polypeptides

Vitamin K-dependent polypeptides as one group of the therapeuticpolypeptides are polypeptides that are γ-carboxylated enzymatically inthe liver using vitamin K as a cofactor. Such vitamin K-dependentpolypeptides e.g. include Factors II, VII, IX, X, Protein C, Protein S,GAS6, and Protein Z.

Human FIX

Human FIX, one member of the group of vitamin K-dependent polypeptides,is a single-chain glycoprotein with a molecular weight of 57 kDa, whichis secreted by liver cells into the blood stream as an inactive zymogenof 415 amino acids. It contains 12 γ-carboxy-glutamic acid residueslocalized in the N-terminal Gla-domain of the polypeptide. The Glaresidues require vitamin K for their biosynthesis. Following the Gladomain there are two epidermal growth factor domains, an activationpeptide, and a trypsin-type serine protease domain. Furtherposttranslational modifications of FIX encompass hydroxylation (Asp 64),N-(Asn157 and Asn167) as well as 0-type glycosylation (Ser53, Ser61,Thr159, Thr169, and Thr172), sulfation (Tyr155), and phosphorylation(Ser158). FIX is converted to its active form, Factor IXa, byproteolysis of the activation peptide at Arg145-Ala146 and Arg180-Val181leading to the formation of two polypeptide chains, an N-terminal lightchain (18 kDa) and a C-terminal heavy chain (28 kDa), which are heldtogether by one disulfide bridge. Activation cleavage of Factor IX canbe achieved in vitro e.g. by Factor XIa or Factor VIIa/TF. Factor IX ispresent in human plasma in a concentration of 5-10 μg/ml. Terminalplasma half-life of Factor IX in humans was found to be about 15 to 18hours (White G C et al. 1997. Recombinant factor IX. Thromb Haemost. 78:261-265; Ewenstein B M et al. 2002. Pharmacokinetic analysis ofplasma-derived and recombinant F IX concentrates in previously treatedpatients with moderate or severe hemophilia B. Transfusion 42:190-197).

Hemophilia B is caused by non-functional or missing Factor IX and istreated with Factor IX concentrates from plasma or a recombinant form ofFactor IX. As haemophilia B patients often receive at least biweeklyprophylactic administrations of Factor IX to avoid spontaneousbleedings, it is desirable to increase the intervals of time betweenadministrations by increasing the half-life of the Factor IX productapplied. An improvement in plasma half-life may bring significantbenefit to the patient. Up to now no pharmaceutical preparation of aFactor IX with improved plasma half-life is commercially available norhave any data been published showing F IX variants with prolonged invivo half-life and almost unchanged molar specific activity incoagulation-related assays. Therefore, a great medical need still existsto develop forms of Factor IX which have a longer functional half-lifein vivo.

Factor VII and Factor VIIa

FVII is a single-chain glycoprotein with a molecular weight of 50 kDa,which is secreted by liver cells into the blood stream as an inactivezymogen of 406 amino acids. FVII is converted to its active form FactorVIIa, by proteolysis of the single peptide bond at Arg152-Ile153 leadingto the formation of two polypeptide chains, a N-terminal light chain (24kDa) and a C-terminal heavy chain (28 kDa), which are held together byone disulfide bridge. In contrast to other vitamin K-dependentcoagulation factors, no activation peptide is cleaved off duringactivation. Activation cleavage of Factor VII can be achieved in vitro,for example, by Factor Xa, Factor IXa, Factor VIIa, Factor XIIa, FactorSeven Activating Protease (FSAP), and thrombin. Mollerup et al.(Biotechnol. Bioeng. (1995) 48: 501-505) reported that some cleavagealso occurs in the heavy chain at Arg290 and/or Arg315.

Factor VII is present in plasma in a concentration of 500 ng/ml. About1% or 5 ng/ml of Factor VII is present as activated Factor VIIa. Theterminal plasma half-life of Factor VII was found to be about 4 hoursand that of Factor VIIa about 2 hours.

By administering supraphysiological concentrations of Factor VIIahemostasis can be achieved bypassing the need for Factor Villa andFactor IXa. The cloning of the cDNA for Factor VII (U.S. Pat. No.4,784,950) made it possible to develop activated Factor VII as apharmaceutical. Factor VIIa was successfully administered for the firsttime in 1988. Ever since the number of indications of Factor VIIa hasgrown steadily showing a potential to become an universal hemostaticagent to stop bleeding (Erhardtsen, 2002). However, the short terminalhalf-life of Factor VIIa of approximately 2 hours and reduced in vivorecovery may be limiting its application. Therefore, a great medicalneed still exists to develop forms of Factor VIIa which have an improvedhalf-life but otherwise almost uncompromised molar specific activity,inactivation kinetics, and/or elimination kinetics after start ofcoagulation.

Therapeutic Fusion Proteins

“Therapeutic fusion proteins” or “fusion proteins” as used herein arecoagulation factors fused to a half-life enhancing polypeptide suchthat, upon administration to a human or other animal, may produce aprophylactic or therapeutic effect. These therapeutic fusion proteinsmay be administered to a human or other animal via, for example,intravenous, intramuscular, subcutaneous, oral, topical, parenteral orother routes. In addition, gene therapy protocols may be used whichinvolve administration of a polynucleotide encoding the fusion proteinor a composition comprising that polynucleotide, such as a plasmid orvector or host cell. Specific classes of therapeutic fusion proteinsdescribed, i.e. by the examples below, are coagulation factors, such asvitamin K-dependent polypeptides, linked to half-life enhancingpolypeptides, such as albumin and immunoglobulin. The expression“therapeutic fusion protein” is used interchangeably with “fusionprotein”.

Half-Life Enhancing Polypeptide (HLEP)

Albumin and immunoglobulin have been described above as examples ofhalf-life enhancing polypeptides (HLEPs). The terms “human serumalbumin” (HSA) and “human albumin” (HA) are used interchangeably in thisapplication. The terms “albumin” and “serum albumin” are broader, andencompass human serum albumin as well as albumin from other species, andfragments and variants thereof. “Albumin,” as used herein, includespolypeptides of the albumin family of proteins. As used herein,“albumin” refers collectively to albumin polypeptide or amino acidsequence, or an albumin fragment or variant having one or morefunctional activities (e.g., biological activities) of albumin. Examplesinclude human serum albumin and bovine serum albumin, including variantsand derivatives thereof, such as genetically engineered or chemicallymodified albumin variants and fragments of albumin proteins. Forexample, “albumin” refers to human albumin or fragments thereof, such asthe mature form of human albumin as shown in SEQ ID NO:1 herein oralbumin from other vertebrates or fragments thereof, or analogs orvariants of these molecules or fragments thereof.

The albumin portion of the albumin fusion proteins may comprise the fulllength of, for instance, the HA sequence as described above, or mayinclude one or more fragments thereof that are capable of stabilizing orprolonging the therapeutic activity. Such fragments may be of 10 or moreamino acids in length or may include about 15, 20, 25, 30, 50, or morecontiguous amino acids from the HA sequence or may include part or allof specific domains of HA.

The albumin portion of the albumin fusion proteins of the invention maycomprise a variant or derivative or analog of normal HA, either naturalor artificial. The therapeutic polypeptide portion of the fusionproteins of the invention may also comprise variants of thecorresponding therapeutic polypeptides as described herein.

The terms “variants” “derivatives” and “analogs”, throughout thisapplication, when applied to any protein disclosed herein, each include,for example, insertions, deletions, and substitutions, eitherconservative or non-conservative, either natural or artificial (i.e.engineered), where such changes do not substantially alter the activesite, or a fragment, such as an active domain that confers thetherapeutic activities of the therapeutic polypeptides, or chemicalmodifications that also allow for therapeutic activity. Examplesinclude, for instance coagulation factors, albumin, or immunoglobulinsthat are 80%, 85%, 90%, and 95% identical in sequence to a wild-type,human coagulation factor, albumin, or immunoglobulin sequence.

In particular, the albumin fusion proteins of the invention may includenaturally occurring polymorphic variants of human albumin and fragmentsof human albumin. The albumin may be derived from any vertebrate,especially any mammal, for example human, cow, sheep, or pig.Non-mammalian albumins include, but are not limited to, hen and salmon.The albumin portion of the albumin-linked polypeptide may be from adifferent animal than the therapeutic polypeptide portion.

Generally speaking, an albumin fragment or variant encompassed withinthe term “albumin” will be at least 10, for example at least 40, or morethan 70 amino acids long. That albumin variant may comprise at least onewhole domain of albumin or fragments of said domains, for exampledomains 1 (amino acids 1-194 of SEQ ID NO:1), 2 (amino acids 195-387 ofSEQ ID NO:1), 3 (amino acids 388-585 of SEQ ID NO:1), 1+2 (1-387 of SEQID NO:1), 2+3 (195-585 of SEQ ID NO:1) or 1+3 (amino acids 1-194 of SEQID NO:1+amino acids 388-585 of SEQ ID NO:1). Each domain is itself madeup of two homologous subdomains namely 1-105, 120-194, 195-291, 316-387,388-491 and 512-585, with flexible inter-subdomain linker regionscomprising residues Lys106 to Glu119, Glu292 to Val315, and Glu492 toAla511.

The albumin portion of an albumin fusion protein of the invention maycomprise at least one subdomain or domain of HA or conservativemodifications thereof.

All fragments and variants of albumin are encompassed by the inventionas fusion partners of a coagulation factor as long as they lead to ahalf-life extension of the therapeutic fusion protein in plasma of atleast 25% as compared to the non-fused coagulation factor.

The albumin family of proteins, included within the term “albumin” usedherein, comprise evolutionarily related serum transport proteins, forexample, albumin, alpha-fetoprotein (AFP; Beattie & Dugaiczyk 1982. Gene20:415-422), afamin (AFM; Lichenstein et al. 1994. J. Biol. Chem.269:18149-18154) and vitamin D binding protein (DBP; Cooke & David 1985.J. Clin. Invest. 76:2420-2424). Alpha-fetoprotein has been claimed toenhance the half-life of an attached therapeutic polypeptide in vivo (WO2005/024044). Their genes represent a multigene cluster with structuraland functional similarities mapping to the same chromosomal region inhumans, mice and rat. The structural similarity of those albumin familymembers suggests their usability as HLEPs. Some embodiments of theinvention, therefore, may use such albumin family members, or fragmentsand variants thereof as defined above, as HLEPs.

Albumin family members encompassed within the term “albumin” herein alsocomprise the full length of the respective proteins AFP, AFM and DBP, ormay include one or more fragments thereof that are capable ofstabilizing or prolonging the therapeutic activity. Such fragments maybe of 10 or more amino acids in length or may include about 15, 20, 25,30, 50, or more contiguous amino acids of the respective proteinsequence or may include part or all of specific domains of therespective protein, as long as the HLEP fragments provide a half-lifeextension of at least 25% as compared to the non-fused coagulationfactor. Albumin family members of the therapeutic fusion proteins of theinvention may also include naturally occurring polymorphic variants ofAFP, AFM and DBP.

The term “immunoglobulin” as used herein, encompasses, for instance, IgGand IgG-fragments, which may also be used as HLEPs, as long as thecorresponding HLEP fragments provide a half-life extension of at least25% as compared to the non-fused coagulation factor. The therapeuticpolypeptide portion may be connected to the IgG or the IgG fragments viaa cleavable linker that allows high molar specific activities of thefusion protein. For example, fusion proteins comprising FVII or VIIa andIgG or fragments thereof may be prepared. A linker sequence of thepresent invention liberating FVII (FVIIa) molecules upon cleavage by aprotease of the coagulation cascade such as, e.g., FXIa, FXa, or FIXacould be able to elevate the clotting activity of the constructs to anactivity level comparable to the monomer/dimer or even higher. A FIX-Fcfusion protein with cleavable linker is exemplarily shown in SEQ IDNO:93. Cleavable linkers such as those shown in table 3a and 3b may alsobe applied in such embodiments.

The invention also relates to fusion proteins comprising linking acoagulation factor, including, for example, a fragment or variantthereof, to the N- or C-terminus of a HLEP, including a fragment orvariant thereof, such that an intervening cleavable peptide linker isintroduced between the therapeutic polypeptide and the HLEP such thatthe fusion protein formed has an increased in vivo half-life compared tothe coagulation factor which has not been linked to a HLEP and that thefusion protein has an at least 25% higher molar specific activitycompared to the corresponding fusion protein with non-cleavable linkerin at least one of the different coagulation-related assays available.

“Coagulation factor” as used in this application includes, but is notlimited to, polypeptides consisting of Factor IX, Factor VII, FactorVIII, von Willebrand Factor, Factor V, Factor X, Factor XI, Factor XII,Factor XIII, Factor I, Factor II (Prothrombin), Protein C, Protein S,GAS6, or Protein Z as well as their activated forms. A “coagulationfactor,” as used herein, also includes variants or derivatives thereof,such as genetically engineered or chemically modified variants or activefragments thereof. For instance, useful therapeutic polypeptides may bewild-type polypeptides or may contain mutations. Degree and location ofglycosylation or other post-translation modifications may vary dependingon the chosen host cells and the nature of the host cellularenvironment. When referring to specific amino acid sequences,posttranslational modifications of such sequences are encompassed inthis application.

“Coagulation factor” within the above definition includes polypeptidesthat have the natural amino acid sequence including any naturalpolymorphisms. It also includes polypeptides with a slightly modifiedamino acid sequence, for instance, a modified N-terminal or C-terminalend including terminal amino acid deletions or additions, as long asthose polypeptides substantially retain the activity of the respectivetherapeutic polypeptide. Variants included may differ in one or moreamino acid residues from the wild type sequence. Examples of suchdifferences may include truncation of the N- and/or C-terminus by one ormore amino acid residues (e.g. 1 to 30 amino acid residues), or additionof one or more extra residues at the N- and/or C-terminus, as well asconservative amino acid substitutions, i.e. substitutions performedwithin groups of amino acids with similar characteristics, e.g. (1)small amino acids, (2) acidic amino acids, (3) polar amino acids, (4)basic amino acids, (5) hydrophobic amino acids, and (6) aromatic aminoacids. Examples of such conservative substitutions are shown in thefollowing table.

TABLE 1 (1) Alanine Glycine (2) Aspartic acid Glutamic acid (3a)Asparagine Glutamine (3b) Serine Threonine (4) Arginine Histidine Lysine(5) Isoleucine Leucine Methionine Valine (6) Phenylalanine TyrosineTryptophane

The in vivo half-life of the fusion proteins of the invention, ingeneral determined as terminal half-life or β-half-life, may be at leastabout 25%, at least about 50%, or more than 100% higher than the in vivohalf-life of the non-fused polypeptide.

The fusion proteins of the present invention also have at least a 25%,and may have at least a 50% or at least 100% increased molar specificactivity compared to the corresponding fusion proteins without cleavablelinkers.

The “molar specific activity” (or molar specific coagulation-relatedactivity as considered here in particular) in this regard is defined forpurposes herein as the activity expressed per mole (or e.g. nmole) ofthe therapeutic polypeptide or therapeutic fusion protein of interest.Calculation of the molar specific activity allows a direct comparison ofthe activity of the different constructs that is not affected by thedifferent molecular weights or optical densities of the polypeptidesstudied. The molar specific activity may be calculated as exemplified intable 2 below for FIX and a FIX-HSA fusion protein.

TABLE 2 Calculation of molar specific activity as shown for a purifiedFIX-HSA fusion protein Molar optical Calculation of molarActivity/Vol/OD₂₈₀ density (OD₍₂₈₀₎ specific activity ProductOD_((280 nm. 1%)) MW (IU/L/OD₂₈₀) at 1 mol/L) (IU/mol) FIX 13.3¹⁾ 57 000determined for 75810 (=MW × =(Activity/Vol/OD₂₈₀) × productOD_((280. 1%))/10) (OD₂₈₀ at 1 mol/L) HSA 5.7²⁾ 66 300 37791 (=MW ×OD_((280. 1%))/10) FIX-HSA determined for 113601 (=sum=(Activity/Vol/OD₂₈₀) × product of molar optical (OD₂₈₀ at 1 mol/L)density of FIX and HSA) ¹⁾R. G. Di Scipio et al., Biochem. 16: 698-706(1977) ²⁾C. Chaudhury et al, J. Exp. Med. 197(3): 315-322 (2003)

In order to determine a molar specific coagulation-related activity, anyassay may be used that determines enzymatic or cofactor activities thatare relevant to the coagulation process.

Therefore “coagulation-related assays” in the sense of the inventioncomprise any assay which determines enzymatic or cofactor activitiesthat are of relevance in the coagulation process or that are able todetermine that either the intrinsic or the extrinsic coagulation cascadehas been activated. The “coagulation-related” assay thus may be directcoagulation assays like aPTT, PT, or the thrombin generation assays.However, other assays such as chromogenic assays applied for specificcoagulation factors are also included. Examples of such assays orcorresponding reagents are Pathromtin® SL (aPTT assay, Dade Behring) orThromborel® S (Prothrombin time assay, Dade Behring) with correspondingcoagulation factor deficient plasma (Dade Behring), Thrombin generationassay kits (Technoclone™, Thrombinoscope™) using e.g. coagulation factordeficient plasma, chromogenic assays like Biophen™ Factor IX (HyphenBioMed), Staclot® FVIIa-rTF (Roche Diagnostics GmbH), Coatest® FactorVIII:C/4 (Chromogenix), or others.

For purposes of this invention, an increase in any one of the aboveassays or an equivalent coagulation-related assay is considered to showan increase in molar specific activity. For example, a 25% increaserefers to a 25% increase in any of the above or an equivalent assay.

To determine whether therapeutic fusion proteins fall within the scopeof the present invention, the standard against which the molar specificactivity of these proteins is compared is a construct in which therespective coagulation factor and the respective HLEP are linked by anon-cleavable linker having the amino acid sequence GGGGGGV.

In the case of FIX, aPTT assays are often used for determination ofcoagulation activity. Such a coagulation assay (aPTT assay) is describedin example 5 in more detail. However, other coagulation-related assaysor assay principles may be applied to determine molar specific activityfor FIX.

Recombinant therapeutic polypeptide drugs are usually expensive and notall countries can afford costly therapies based on such drugs.Increasing the in vivo recovery of such drugs could make the use ofthese products cheaper and subsequently more patients would benefit fromthem. In the case of the fusion proteins of the present invention anincreased in vivo recovery would also be a desirable advantage. “In vivorecovery” as used herein in the sense of the invention means the amountof product found in blood or plasma shortly after administration of theproduct. For detection of the in vivo recovery in general the plasmacontent is determined a few minutes (e.g. 5 or 15 min) afteradministration of the product.

Although it is desirable to have a high in vivo recovery and a longhalf-life for a non-activated coagulation factor, it is advantageous tolimit the half-life of a coagulation factor after its activation or theactivation of its co-factor in order to avoid a prothrombotic risk.Therefore, after the coagulation process has been initiated, thehalf-life of the active coagulation factor should again be reduced. Thiscan either be achieved by enhancing inactivation in acoagulation-related mode or by elimination of the coagulation factor.

“Inactivation” according to the present invention means the decrease ofactivity of the therapeutic polypeptide which can be caused, forexample, by a complex formation of a coagulation factor and an inhibitorof the corresponding coagulation factor or by further proteolyticcleavage as known, e.g., in the case of FVIII and FV.

The “inactivation rate” of an activated therapeutic fusion protein isdefined as the rate the activity is declining, e.g., by reaction withinhibitors or by proteolytic inactivation. The inactivation rate may bemeasured by following the molar specific activity of the activatedcoagulation factor over time in the presence of physiologic amounts ofinhibitors of this coagulation factor. Alternatively, the inactivationrate may be determined after administration of the activated product toan animal followed by testing of plasma samples at an appropriate timeframe using activity and antigen assays.

To determine whether therapeutic fusion proteins fall within the scopeof the present invention, the standard against which the molar specificactivity of these proteins is compared is a construct in which therespective coagulation factor and the respective HLEP are linked by anon-cleavable linker having the amino acid sequence GGGGGGV.

The “elimination rate” of an activated therapeutic fusion protein isdefined as the rate the polypeptide is eliminated from the circulationof humans or other animals. The elimination rate may be determined bymeasuring the pharmacokinetics of the activated, therapeutic fusionprotein after intravenous administration. Using an antigen assay, theelimination by direct removal from the circulation can be determined.Using an activity assay in addition, a specific removal and inactivationrate may be determined.

To determine whether therapeutic fusion proteins fall within the scopeof the present invention, the standard against which the molar specificactivity of these proteins is compared is a construct in which therespective coagulation factor and the respective HLEP are linked by anon-cleavable linker having the amino acid sequence GGGGGGV.

According to some embodiments of this invention, the therapeuticpolypeptide moiety is coupled to the HLEP moiety by a cleavable peptidelinker. The linker should be non-immunogenic and should be flexibleenough to allow cleavage by proteases. The cleavage of the linker shouldproceed comparably fast as the activation of the therapeutic polypeptidewithin the fusion protein, if the fusion protein is a zymogen.

The cleavable linker may comprise a sequence derived from

-   -   a) the therapeutic polypeptide to be administered itself if it        contains proteolytic cleavage sites that are proteolytically        cleaved during activation of the therapeutic polypeptide,    -   b) a substrate polypeptide of this therapeutic polypeptide, or    -   c) a substrate polypeptide cleaved by a protease which is        activated or formed by the direct or indirect involvement of the        therapeutic polypeptide.

The peptide linker in some embodiments comprises a sequence of thetherapeutic polypeptide to be applied, which should result in adecreased risk of neoantigenic properties of the expressed fusionprotein. Also in case the therapeutic protein is a zymogen (e.g. needsto be proteolytically activated) the kinetics of the peptide linkercleavage will more closely reflect the coagulation-related activationkinetics of the zymogen in some embodiments.

In some embodiments, the therapeutic polypeptide is FIX zymogen and theHLEP is albumin. In some of those embodiments the linker sequence iseither derived from the sequences of the activation regions of FIX, fromthe cleavage region of any substrate of FIX like FX or FVII or from thecleavage region of any substrate polypeptide that is cleaved by aprotease in whose activation FIXa is involved.

In yet other embodiments, the linker peptide is derived from FIX itself.In other embodiments the linker peptide is derived from FX or FVII. Inother embodiments the linker sequence comprises two cleavage sequencesthat can be cleaved by FXIa or FVIIa/TF, two physiologically relevantactivators of FIX.

Exemplary combinations of therapeutic polypeptide, peptide linker, andHLEP include the constructs listed in tables 3a and 3b, but are notlimited to them:

TABLE 3A Examples of possible constructs Linker derived from (withCoagulation modifications, SEQ factor Linker HLEP if applicable) ID NO:Linker not cleavable or not sufficiently rapidly cleavable FIX HSA FIXRI HSA FIX GGGGGGV (Sheffield et al.) HSA 94 FIX (GGS)_(n)GS HSA FIXSS(GGS)₇GS HSA 30 FIX SSNGS(GGS)₃NGS(GGS)₃GGNGS HSA 31Linker with one cleavage site FIX (1-412) SVSQTSKLTRAETVFPDVD HSA FIX 36FIX (1-412) SVSQTSKLTRAETVFPDVDGS HSA FIX 37 FIX SVSQTSKLTRAETVFPDVD HSAFIX 38 FIX SVSQTSKLTRAETVFPDVDGSGGS HSA FIX 95 FIX SVSQTSKLTRAETVFPDVDGSHSA FIX 39 FIX SVSIDTSKLTRAETVFPDVDNGS HSA FIX 40 FIX SVSQTSKLTRAETVFPDVHSA FIX 96 FIX QTSKLTRAETVFPDV HSA FIX 97 FIX SKLTRAETVFPDV HSA FIX 98FIX SVSQTSKLTRAETVFP HSA FIX 99 FIX SVSQTSKLTRAETVF HSA FIX 100 FIXQTSKLTRAETVF HSA FIX 101 FIX SKLTRAETVF HSA FIX 102 FIX SVSQTSKLTRAETHSA FIX 103 FIX QTSKLTRAET HSA FIX 104 FIX SKLTRAET HSA FIX 105 FIXSVSQTSKLTRGETVFPDVD HSA FIX 41 FIX SVSQTSKLTRTETVFPDVD HSA FIX 42 FIXSVSQTSKLTRSETVFPDVD HSA FIX 43 FIX SVSQTSKLTRLETVFPDVD HSA FIX 44 FIXSVSQTSKLTRTEAVFPDVD HSA FIX 45 FIX SVSQTSKLTRGEAVFPDVD HSA FIX 46 FIXQTSKLTRAETVFPDVDGS HSA FIX 106 FIX SKLTRAETVFPDVDGS NSA FIX 107 FIXSKLTRAETVFPDVD HSA FIX 47 FIX QSFNDFTRVVGGED HSA FIX 48 FIXQSFNDFTRVVGGEDGS HSA FIX 49 FIX QSFNDFTRVVGGE HSA FIX 108 FIXQSFNDFTRTVGGED HSA FIX 50 FIX QSFNDFTRLVGGED HSA FIX 51 FIXQSFNDFTRGVGGED HSA FIX 52 FIX QSFNDFTRVVGGEDNGS HSA FIX 53 FIXQSFNDFTRVVGGEDN HSA FIX 54 FIX PERGDNNLTRIVGGQEGS HSA FX 109 FIXPERGDNNLTRIVGGQE HSA FX 61 FIX PERGDNNLTRIVGGQ HSA FX 110 FIXDNNLTRIVGGQ HSA FX 111 FIX SVSQTSKLTRAETVFPDVD Fc FIX 62 FIXQSFNDFTRVVGGEDN Fc FIX 63 FIX (1-412) SVSQTSKLTRAETVFPDVD Fc FIX 64 FIXASKPQGRIVGG HSAdelDAH FVII 112 FIX KRNASKPQGRIVGGKV HSA FVII 65 FIXPEEPQLRMKNNEEAED HSA FVIII 66 FIX DNSPSFIQIRSVAKKHPKT HSA FVIII 67 FIXLSKNNAIEPRSFSQNSRHPS HSA FVIII 68 FIX DEDENQSPRSFQKKTRHYFIA HSA FVIII 69FIX SPHVLRNRAQSGSVPQ HSA FVIII 70 FVII or FVIIa PEEPQLRMKNNEEAEDYDDDLTDSHSA FVIII 71 FVII or FVIIa DDDNSPSFIQIRSVAKKHPKTWVHYAAEEED HSA FVIII 72FVII or FVIIa LSKNNAIEPRSFSQNSRHPSTRQKQFNA HSA FVIII 73 FVII or FVIIaDEDENQSPRSFQKKTRHYFIAA HSA FVIII 74 FVII or FVIIaDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFT HSA FVIII 75 FVIIIDerived from cleavage sites HSA FVIII, FIX orof FVIII, FIX, or Fibrinogen Fgn VWF Derived from cleavage sites of VWF,HSA FIX, FVIII, VWF FVIII, or FIX VWF DIYDEDENQSPRSFQKKTRHYFIA HSA FVIII76 VWF DNSPSFIQIRSVAKKHP HSA FVIII 77 VWF LSKNNAIEPRSFSQNSRHPS HSA FVIII78 FIX PVSQTSKLTRAETVFPDV HSA FIX 113 FIX PSVSQTSKLTRAETVFPDV HSA FIX114

In the case of linkers derived from the N-terminal region of the FIXactivation peptide, according to the natural polymorphism T148-A148 thesequences may also contain A instead of T at this position.

TABLE 3B Examples of possible constructs with two or more cleavage sitesCoagulation Linker derived from (par- SEQ factor Linker HLEPtially incl. Modifications) ID NO: Linker with two cleavage sites FIXSVSOTSKLTRAETVFPDVTQPERGDNNLTRI HSA FIX, FX 79 VGGQE FIXSKLTRAETVFPDNNLTRIVGGQE HSA FIX, FX 80 FIX RAETVFPDVTQPERGDNNLTRIVGGQEHSA FIX, FX 81 FIX RAETVFPERGDNNLTRIVGGQE HSA FIX, FX 82 FIXSVSQTSKLTRAETVFPDVDYVNNLTRIVGGQE HSA FIX, FX 83 FIXSVSQTSKLTRAETVFPDVDNNLTRIVGGQE HSA FIX, FX 84 FIXSVSQTSKLTRAETVFPDVDNNLTRIVGGQE HSA FIX, FX 85 FIXSVSQTSKLTRAETVFPDVDYVNSTEAETILDNITQ HSA FIX 86 STQSFNDFTRVVGGEDA FIXSVSQTSKLTRAETVFPDVQSFNDFTRVVGGED HSA FIX 87 FIXSVSQTSKLTRAETVFPDVDSFNDFTRVVGGED HSA FIX 88 FIXSVSQTSKLTRAETVFPDVNASKPQGRIVGGKV HSA FIX and FVII 89 FIXSVSQTSKLTRAETVFPDVNASKPQGRLVGGKV HSA FIX and FVII 90 FIXSVSQTSKLTRAETVFPDVNASKPQGRTVGGKV HSA FIX and FVII 91 FIXSVSQTSKLTRAETVFPDVD Fc 92

Variants and fragments of the linkers described in tables 3a and 3b arealso encompassed in the present invention as long as the linkers canstill be cleaved by the protease or the proteases that cleave thelinkers of tables 3a and 3b or by the type of proteases defined above.

Other combinations of the cleavage sequences described above and theirvariants shall be included in the present invention.

In another embodiment, amino acid substitutions are included that changethe posttranslational modification pattern of the peptide linker. Thesecan be, for example, substitutions of amino acids that are glycosylated,sulphated, or phosphorylated.

In another embodiment of the invention the peptide linker between thetherapeutic polypeptide and the HLEP moiety contains consensus sites forthe addition of posttranslational modifications. Such modifications maycomprise glycosylation sites. For example, such modifications maycomprise at least one N-glycosylation site of the structureAsn-X-Ser/Thr, wherein X denotes any amino acid except proline.Furthermore, such N-glycosylation sites may be inserted close to theamino and/or carboxy terminus of the peptide linker such that they arecapable of shielding potential neo-epitopes which might develop at thesequences where the therapeutic polypeptide moiety is transitioning intothe peptide linker or where the peptide linker is transitioning into,for example, an albumin moiety sequence.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: In vitro activation of FIX-albumin fusion proteins by FXIa at37° C. at a molar ratio of FXIa to fusion protein of about 1:500. Onefusion protein with non-cleavable linker (1478/797) and two fusionproteins with cleavable linker (1088/797 and 1089/797) were used.Samples were analyzed by SDS-PAGE under reducing conditions followed byCoomassie blue staining

FIG. 2: Pharmacokinetics of activated rec FIX and FIX-albumin fusionproteins with and without cleavable linker in comparison tonon-activated fusion proteins.

FIG. 3: Inactivation of activated rec FIX or FIX-albumin fusion proteinby AT. Residual FIX activity was determined after 120 min using anon-activated partial thromboplastin time assay.

EXAMPLES Example 1 Generation of cDNAs Encoding FIX and FIX-AlbuminFusion Proteins

Factor IX coding sequence was amplified by PCR from a human liver cDNAlibrary (ProQuest, Invitrogen) using primers We1403 and We1404 (SEQ IDNOS:5 and 6). After a second round of PCR using primers We1405 andWe1406 (SEQ ID NOS:7 and 8) the resulting fragment was cloned intopCR4TOPO (Invitrogen). From there the FIX cDNA was transferred as anEcoRI Fragment into the EcoRI site of pIRESpuro3 (BD Biosciences)wherein an internal XhoI site had been deleted previously. The resultingplasmid was designated pFIX-496 and was the expression vector for factorIX wild-type.

For the generation of albumin fusion constructs the FIX cDNA wasreamplified by PCR under standard conditions using primers We2610 andWe2611 (SEQ ID NOS:9 and 10) deleting the stop codon and introducing anXhoI site instead. The resulting FIX fragment was digested withrestriction endonucleases EcoRI and XhoI and ligated into an EcoRI/BamH1digested pIRESpuro3 together with one XhoI/BamH1 digested linkerfragment as described below.

Two different glycine/serine linker fragments without internal cleavagesites were generated: Oligonucleotides We2148 and We2150 (SEQ ID NOS:11and 12) were annealed in equimolar concentrations (10 pmol) understandard PCR conditions, filled up and amplified using a PCR protocol ofa 2 min. initial denaturation at 94° C. followed by 7 cycles of 15 sec.of denaturation at 94° C., 15 sec. of annealing at 55° C. and 15 sec. ofelongation at 72° C., and finalized by an extension step of 5 min at 72°C. The same procedure was performed using oligonucleotides We2156 andWe2157 (SEQ ID NOS:13 and 14). The resulting linker fragments weredigested with restriction endonucleases XhoI and BamH1 and usedseparately in the above described ligation reaction. The resultingplasmids therefore contained the coding sequence for FIX and aC-terminal extension of a glycine/serine linker.

Two different cleavable linker fragments derived from the activationsites of FIX were generated: Oligonucleotides We2335 and We2336 (SEQ IDNOS:15 and 16), containing the activation cleavage site of the FIX lightchain/activation peptide border region, were annealed, filled, andamplified as described above. The resulting linker fragment was digestedwith restriction endonucleases XhoI and BamH1 and used in the abovedescribed ligation reaction. The resulting plasmid therefore containedthe coding sequence for FIX and a C-terminal extension of a cleavableFIX sequence (amino acids 136 to 154 of SEQ ID NO:2). In a subsequentsite directed mutagenesis reaction with a commercially availablemutagenesis kit (QuickChange XL Site Directed Mutagenesis Kit,Stratagene) using oligonucleotides We2636 and We2637 (SEQ ID NOS:17 and18) the XhoI site was deleted.

For generation of the second cleavable linker fragment derived from FIX,the same procedure was performed using oligonucleotides We2337 andWe2338 (SEQ ID NOS:19 and 20) for linker construction. The resultinglinker fragment was digested with restriction endonucleases XhoI andBamH1 and used in the above described ligation reaction. The resultingplasmid now contained the coding sequence for FIX and a C-terminalextension of a cleavable FIX sequence derived from the activationcleavage site of the FIX activation peptide/heavy chain border region(amino acids 173 to 186 of SEQ ID NO:2). Oligonucleotides We2638 and We2639 (SEQ ID NOS:21 and 22) were used for deletion of the XhoI site asdescribed above.

In the next cloning step the above generated plasmids were digested withBamH1 and a BamH1 fragment containing the cDNA of mature human albuminwas inserted. This fragment had been generated by PCR on an albumin cDNAsequence using primers We1862 and We1902 (SEQ ID NOS:23 and 24) understandard conditions.

The final plasmids with non-cleavable glycine/serine linkers weredesignated pFIX-980 (SEQ ID NO:30) and pFIX-986 (SEQ ID NO:31),respectively. The final plasmids with cleavable linkers derived from FIXsequences were designated pFIX-1088 (SEQ ID NO:40) and pFIX-1089 (SEQ IDNO:49), respectively. Their linker sequences and the C-terminal FIX andN-terminal albumin sequences are outlined below. Proteolytic cleavagesites within the linkers are indicated with arrows, the FIX derivedlinker sequences are underlined.

For expression in CHO cells the coding sequences for the FIX albuminfusion protein were transferred into vectors pIRESneo3 (BD Biosciences)or pcDNA3.1 (Invitrogen), respectively.

Using the above protocols and plasmids and by applying molecular biologytechniques known to those skilled in the art (and as described e.g. inCurrent Protocols in Molecular Biology, Ausubel F M et al. (eds.),including Supplement 80, published October 2007, John Wiley & Sons,Inc.; http://www.currentprotocols. com/WileyCDA/) other constructs canbe made with insertions of different linker sequences, e.g. as describedin tables 3a and 3b.

For efficient processing of the propeptide in cells expressing FIX inhigh amounts coexpression of furin is required (Wasley L C et al. 1993.PACE/Furin can process the vitamin K-dependent pro-factor IX precursorwithin the secretory pathway. J. Biol. Chem. 268:8458-8465). Furin wasamplified from a liver cDNA library (Ambion) using primers We1791 andWe1792 (SEQ ID NOS:25 and 26). A second round of PCR using primersWe1808 and We1809 (SEQ ID NOS:27 and 28) yielded a furin fragment wherethe carboxyterminal transmembrane domain (TM) was deleted and a stopcodon introduced; this fragment was cloned into pCR4TOPO (Invitrogen).From there the furinΔTM cDNA was transferred as an EcoRI/NotI Fragmentinto the EcoRI/NotI sites of pIRESpuro3 (BD Biosciences) wherein aninternal XhoI site had been deleted previously. The resulting plasmidwas designated pFu-797. This plasmid was cotransfected with all FIXconstructs in a 1:5 (pFu-797:pFIX-xxx) molar ratio. The amino acidsequence of the secreted furin encoded by pFu-797 is given as SEQ-IDNO:29.

Example 2 Transfection and Expression of FIX and FIX-Albumin FusionProteins

Plasmids were grown up in E. coli TOP10 (Invitrogen) and purified usingstandard protocols (Qiagen). HEK-293 cells were transfected using theLipofectamine 2000 reagent (Invitrogen) and grown up in serum-freemedium (Invitrogen 293 Express) in the presence of 50 ng/ml Vitamin Kand 4 μg/ml Puromycin. Transfected cell populations were spread throughT-flasks into roller bottles or small-scale fermenters from whichsupernatants were harvested for purification.

Alternatively, CHO K1 or DG44 cells (Invitrogen) were transfected usingthe Lipofectamine 2000 reagent (Invitrogen) and grown up in serum-freemedium (Invitrogen CD-CHO) in the presence of 50 ng/ml Vitamin K and500-750 ng/ml Geneticin. High expressing clones were selected and spreadthrough T-flasks into roller bottles or small-scale fermenters fromwhich supernatants were harvested for purification.

Example 3 Purification of FIX and FIX-Albumin Fusion Proteins

Cell culture harvest containing FIX or FIX albumin fusion protein wasapplied on a Q-Sepharose FF column previously equilibrated with 50 mMTrisxHCl/100 mM NaCl buffer pH 8.0. Subsequently, the column was washedwith equilibration buffer containing 200 mM NaCl. Elution of the boundFIX or FIX fusion protein was achieved by a salt gradient using 50 mMTrisxHCl/200 mM NaCl buffer pH 8.0 as a basis. The eluate was furtherpurified by column chromatography on a hydroxylapatite resin. For thispurpose, the eluate of the Q-Sepharose FF column was loaded on ahydroxylapatite chromatography column equilibrated with 50 mMTrisxHCl/100 mM NaCl buffer pH 7.2. The column was washed with the samebuffer and FIX or FIX-HSA were eluted using a potassium phosphategradient at pH 7.2. The eluate was dialyzed to reduce the saltconcentration and used for biochemical analysis as well as fordetermination of the pharmacokinetic parameters. FIX antigen andactivity were determined as described in example 5.

Example 4 Alternative Purification Scheme of FIX and FIX-Albumin FusionProteins

As described in example 3, cell culture harvest containing FIX or FIXalbumin fusion protein was purified by chromatography on Q-Sepharose FF.The Q-Sepharose eluate was further purified by chromatography on aHeparin-Fractogel column. For this purpose, the Heparin-Fractogel columnwas equilibrated using 50 mM Tris×HCl, 50 mM NaCl pH 8.0 buffer (EP),the Q-Sepharose FF eluate was applied and the column was washed withequilibration buffer containing 75 mM NaCl. FIX or FIX albumin fusionprotein, respectively, was eluted using EP adjusted to 300 mM NaCl.

The Heparin-Fractogel eluate was further purified by chromatography on ahydroxylapatite chromatography column as described in example 3. Thepurified FIX resp. FIX albumin fusion protein concentrate was subjectedto FIX activity and antigen determination according to example 5 andcharacterized by further in vitro and in vivo investigations.

Example 5 Determination of FIX Activity and Antigen

FIX activity was determined as clotting or coagulation activity (FIX:C)using commercially available aPTT reagents (Pathromtin SL and FIXdepleted plasma, Dade Behring). An internal substandard calibratedagainst the WHO International FIX concentrate Standard (96/854) was usedas a reference.

FIX antigen (FIX:Ag) was determined by an ELISA acc. to standardprotocols known to those skilled in the art. Briefly, microtiter plateswere incubated with 100 μL per well of the capture antibody (Pairedantibodies for FIX ELISA 1:200, Cedarlane, but other sources ofappropriate antibodies may also be applied) overnight at ambienttemperature. After washing plates three times with washing buffer B(Sigma P3563), each well was incubated with 200 μL blocking buffer C(Sigma P3688) for one hour at ambient temperature. After another threewash steps with buffer B, serial dilutions of the test sample in bufferB as well as serial dilutions of a substandard (SHP) in buffer B(volumes per well: 100 μL) were incubated for two hours at ambienttemperature. After three wash steps with buffer B, 100 μL of a 1:200dilution of the detection antibody (Paired antibodies for FIX ELISA,peroxidase labelled, Cedarlane) in buffer B were added to each well andincubated for another two hours at ambient temperature. After three washsteps with buffer B, 100 μL of substrate solution (TMB, Dade Behring,OUVF) were added per well and incubated for 30 minutes at ambienttemperature in the dark. Addition of 100 μL undiluted stop solution(Dade Behring, OSFA) prepared the samples for reading in a suitablemicroplate reader at 450 nm wavelength. Concentrations of test sampleswere then calculated using the standard curve with standard human plasmaas reference.

Example 6 Comparison of FIX-Activity/FIX-Antigen Ratio of DifferentFIX-Albumin Fusion Proteins in Cell Culture Supernatant

Cell culture supernatants of HEK cells transfected with DNA constructscoding for FIX-albumin fusion proteins that contained different linkerpeptides were subjected to FIX activity and antigen testing as describedabove (see example 5). The ratio of FIX:C to FIX:Ag was calculatedrepresenting a measure directly proportional to molar specific activityof the different constructs.

The results shown in table 4 indicate that there is an increase inactivity/antigen ratio upon introduction of cleavable linkers into theFIX-HSA molecule. It also shows that the cleavable linker peptide shouldhave a length of more than two amino acids in order to provide clearlyincreased activity/antigen ratios.

TABLE 4 FIX:C/FIX:Ag ratios of FIX-albumin fusionproteins containing different linker peptides Fold increase comparedto fusion protein 980/797 with non- cleavable FIX-HSA FIX:C/ linkerconstruct Linker FIX:Ag (GGGGGGV) 1182/797 None <0.031 1366/797 RI<0.068 1478/863 GGGGGGV  0.041 -- (Sheffield et al.)  980/797 SS(GGS)₇GS 0.070  1.7  986/797 SSNGS(GGS)3NGS  0.076  1.9 (GGS)3GGNGS 1483/863SVSQTSKLTR AETVFPDVD  0.688 16.8 GSGGS 1088/797 SVSQTSKLTR AETVFPDVD GS 0.832 20.3 1365/797 SVSQTSKLTR AETVFPDVD  0.630 15.4 1482/863SVSQTSKLTR AETVFP  0.482 11.8 1087/797 SVSQTSKLTR AETVFPDVD GS  0.47211.5 (FIX deltaKLT) 1089/797 QSFNDFTR VVGGED GS  0.532 13.0 1091/797PERGDNNLTR IVGGQE GS  0.111  2.7

Example 7 Comparison of FIX and FIX-Albumin Fusion Proteins in Respectto Molar Specific Activity, Terminal In Vivo Half-Life and In VivoRecovery in Rats or Rabbits

Purified recombinant wild type FIX (rFIX 496/797) and FIX-albumin fusionproteins (rFIX 980/797, rFIX 986/797, rFIX-1088/797 and rFIX 1089/797)were tested for FIX activity in a clotting assay as described above. Inparallel, the difference of the optical density at 280 and 320 nm wasdetermined as a measure for protein concentration (OD280-320). Theratios of activity per OD280-320 were calculated and based on the molaroptical densities the molar specific activities were calculated. In thefollowing table 5 the results are summarized.

TABLE 5Molar specific activities of wt FIX compared to FIX-albumin fusionsOptical FIX Molar density clotting Activity/ specific (OD280- activityVol/OD activity* Linker 320) (IU/mL) (IU/mL/OD) (IU/nmol)rFIX.wt (496/797) — 0.3798 21.2 55.8 4.23 rFIX-HSA (non-GGGGGGV (Sheffield et al.) 2.9189 5.8 2.0 0.23 cleavable, 1478/863rFIX-HSA (non- SS(GGS)7GS 1.1122 3.4 3.0 0.35 cleavable, 980/797)rFIX-HSA (non-

S NGS (GGS)3 NGS (GGS)3 0.8107 3.2 4.0 0.45 cleavable, 986/797) GGN G

rFIX-HSA (cleavable, FXIa cleavable 0.3421 11.9 34.8 3.95 1088/797)rFIX-HSA (cleavable, FXIa cleavable 0.4512 11.3 25.0 2.84 1089/797)*Molar specific activity based on activity, optical density and thefollowing molar optical densities: Molar optical density of FIX: OD(280nm, 1 mol/L) = 75 810 Molar optical density of albumin: OD(280 nm, 1mol/L) = 37 791 Molar optical density of FIX-albumin fusion protein:OD(280 nm, 1 mol/L) = 113 601

indicates data missing or illegible when filed

Taking the results summarized in Table 5 into account, two constructsthat were generated according to the present invention show highlyincreased molar specific activities compared to the fusion proteins withnon-cleavable linkers. In addition, the molar specific activity of theseconstructs was only moderately decreased compared to wild type rFIX.

In vitro investigations of the proteolytic cleavage reactions by FactorXIa (FXIa) confirmed that FIX-albumin fusion proteins containing acleavable linker such as construct no. 1088/797 or 1089/797 areactivated and in parallel the linker is cleaved resulting in release ofthe albumin moiety (FIG. 1). The fusion protein with non-cleavablelinker did not show a corresponding release of the albumin moiety.

In the case of FVIIa as cleaving protease in the presence of tissuefactor, the FIX-albumin fusion proteins 1088/797 or 1089/797 containinga cleavable linker also showed release of the albumin moiety in parallelto release of the FIX activation peptide (Data not shown).

In addition to determination of molar specific coagulation activity, thepolypeptides no. 496/797, 980/797, 986/797, 1088/797 and 1089/797described above were administered intravenously to narcotized CD/Lewisrats (6 rats per substance) and/or rabbits (4 rabbits per substance)with a dose of 50 IU/kg body weight. Blood samples were drawn prior totest substance administration and at appropriate intervals starting at 5minutes after administration of the test substances. FIX antigen contentwas subsequently quantified by an ELISA assay specific for human FactorIX (see above). The mean values of the respective groups were used tocalculate in vivo recovery after 5 min. Half-lives for each protein werecalculated using the time points of the beta phase of elimination(terminal half-life) according to the formula t_(1/2)=ln2/k, whereas kis the slope of the regression line obtained upon plotting FIX:Ag levelsin logarithmic scale and time in linear scale.

Calculated in vivo half-lives are summarized in table 6. In rats as wellas in rabbits the in vivo half-lives of the FIX-albumin fusion proteinswere found to be significantly increased in comparison to non-fusedwild-type recombinant FIX prepared in-house or in comparison to thecommercially available recombinant FIX product BeneFIX®. The in vivohalf-lives of the albumin fusion proteins compared to BeneFIX® wereincreased to about 200-400%, depending on the animal species orconstruct used (Table 6).

To evaluate the in vivo recovery, the FIX antigen levels measured per mLof plasma at their maximum concentrations after intravenousadministration (t=5 min) were related to the amount of product appliedper kg. Alternatively, a percentage was calculated by relating thedetermined antigen level (IU/mL) 5 min post infusion to the theoreticalproduct level expected at 100% recovery (product applied per kg dividedby an assumed plasma volume of 40 mL per kg). The in vivo recoveries(IVR) of the FIX-albumin fusion proteins were significantly higher thanthe in vitro recoveries of rFIX (496/797) or BeneFIX® (Table 7).

TABLE 6 Terminal in vivo half-lives of FIX preparations derived fromrecombinant expression (BeneFIX ®, rFIX 496/797) and FIX albumin fusionproteins (rFIX 980/797, rFIX 986/797, rFIX 1088/797, and rFIX 1089/797)after intravenous administration of 50 IU/kg into rats and/or 50 IU/kginto rabbits, respectively. Rat experiments PSR18-05, Rabbit experimentPSR06-05, PSR02-05 PSK11-05 relative to relative to Terminal half-BeneFIX Terminal half- BeneFIX life (h) [%] life (h) [%] rFIX 496/7974.5* 91 n.t. n.t. rFIX 980/797 11.6* 234 36.9° 410 29.3° (2^(nd) exp.)326 rFIX 986/797 10.5* 212 n.t. n.t. rFIX 1088/797 8.3* 168 30.3° 337rFIX 1089/797 10.5* 212 n.t. n.t. BeneFIX 4.95* (mean of 100  9.0° 1005.3 and 4.6) *Determined between 120 and 1440 min °Determined between 4and 96 h

TABLE 7 In vivo recoveries (amount of substance 5 minutes postadministration) of recombinant FIX preparations (BeneFIX, rFIX 496/797)and FIX albumin fusion proteins (rFIX 1088/797, rFIX 1089/797) afterintravenous administration of 50 IU/kg into rats. The percentage of invivo recovery was calculated based on an assumed plasma volume of 40mL/kg. rat experiment in vivo recovery IU/dL per relative to IU/kg/[%]*BeneFIX [%] rFIX 0.462/18.5 74.6 496/797 rFIX 1.034/41.4 166.5 1088/797rFIX 1.063/42.5 171.2 1089/797 BeneFIX 0.621/24.8 100 *Calculated basedon a plasma volume of 40 mL/kg

Example 8 In Vitro Activation of FIX Albumin Fusion Proteinswith/without Cleavable Linker (1088/797 and 980/797) and Determinationof Pharmacokinetics in Rats

FIX-albumin fusion proteins and rec FIX were activated in vitro usingcommercially available Factor XIa (Kordia). Briefly, identical molaramounts of FIX or FIX-albumin fusion protein (3.0×10⁻⁶ mol/L) wereactivated at 37° C. in solution in the presence of FXIa (1.9×10⁻⁸ mol/L)and CaCl₂ (1.5 mmol/L) buffered at pH 6.8. After complete activation asshown by SDS-PAGE the reaction was stopped by addition of a 5× molarexcess of C1-Inhibitor (Berinert® P) based on the amount of FXIa. Thesamples were stored frozen below −70° C. until start of pharmacokineticinvestigation.

A pharmacokinetic investigation of the activated FIX and the FIX-albuminfusion proteins was performed in rats as described in example 7 and theresults were compared to a pharmacokinetic results coveringnon-activated fusion proteins. It turned out that the activated fusionproteins demonstrated significantly reduced half-lives as well as AUC'scompared to the non-activated molecules (FIG. 2). Upon activation theFIX-fusion protein with cleavable linker (1088/797) showed apharmacokinetic behaviour very similar to activated rec FIX (BeneFIX®)whereas the activated fusion protein with non-cleavable linker (980/797)resulted in a higher initial as well as terminal half-life compared toactivated fusion protein 1088/797 with cleavable linker. Therefore, inthis example, the cleavable linker results in increased elimination ofthe coagulation factor after activation and, therefore, avoidsaccumulation of potentially thrombogenic, activated fusion proteins withextended half-lives.

Example 9 Comparison of FIX-Albumin Fusion Proteins with/withoutCleavable Linker in Respect to Inactivation Rate of the ActivatedCoagulation Factors by Antithrombin III (AT)

FIX fusion proteins with (1088/797) and without (980/797) cleavablelinker were activated by incubation with FXIa as described in example 8.The activated factors were incubated with AT for 120 min and residualFIXa activity was determined using a manual FIX clotting assay methodwithout activation (naPTT, see below) acc. to Schnitger and Gross. Ascontrol samples the activated FIX-albumin fusion proteins were used inpresence of the same amount of AT but without incubation.

The FIX activity was determined with the aid of a non-activated partialthromboplastin time assay (naPTT) using FIX deficient plasma from DadeBehring. The samples were prediluted in a buffer of pH 6.8 containingHis, Gly, Sucrose, and Tween® 80. The whole determination was performedusing coagulometers acc. to Schnitger & Gross. A mixture of 0.1 ml F IXdeficient plasma, 0.1 ml sample, and 0.1 ml of 0.1% Phospholipids(Rhone-Poulenc-Nattermann, 1:3 prediluted in imidazole buffersupplemented with 1% HSA) was incubated for 2 minutes at 37° C. Thecoagulation reaction was initiated by adding 0.1 ml 0.025 mol/l CaCl₂solution and the clotting time was determined.

FIG. 3 shows the results of a corresponding inactivation experiment. Inthe case of the fusion protein with cleavable linker (1088/797) anincrease in clotting time from 210 to 540 sec (factor of 2.57×)demonstrated an accelerated inactivation process of FIXa activity by ATcompared to a fusion protein with non-cleavable linker (980/797) thatonly showed an increase from 196 to 411 sec (factor of 2.10×). Mostprobably, the albumin residue sterically affects the AT dependentinactivation process in the case of the fusion protein withnon-cleavable linker whereas in the case of the fusion protein withcleavable linker the albumin residue is cleaved off resulting in anaccelerated inactivation by AT.

Example 10 Design of FIX-HSA Fusion Proteins with Reduced Immunogenicity

As there is with any fusion between two proteins a slight riskassociated that a neoepitope is created around the fusion point it wasinvestigated whether the linker region as described in table 3a and 3bcould be modified in order to decrease this risk.

In the course of this investigation all proposed linker sequences andthe adjacent regions of FIX and HSA were analyzed for potential T-cellepitopes by way of prediction of binding capabability to multiple MHC-IIalleles. One of these approaches involved the PreDeFT analysis offeredby the company EpiVax (146 Clifford St., Providence, R.I. 02903, USA) inwhich the input sequences were parsed into overlapping 9-mer frameswhere each frame overlaps the last by 8 amino acids. Each frame was thenassessed for its ability to bind with a set of common HLA. Thesedetailed findings were then summarized producing regional and overallassessments of immunogenic potential. Finally, any epitope clustersidentified were screened against the non-redundant protein database atGenBank and EpiVax's own database of known MHC ligands and T-cellepitopes.

As a result of these in silico predictions the following FIX fusionproteins were cloned, expressed and purified:

SEQ ID NO: 113 FIX- PVSQTSKLTRAETVFPDV-HSA SEQ ID NO: 114FIX-PSVSQTSKLTRAETVFPDV-HSA

Example 11 Neoantigenicity Test

FIX-HSA fusion proteins comprising linkers SEQ ID NO:113 or 114 can beshown to display a reduced immunogenicity compared to fusion proteinscomprising different linkers or to display a comparable immunogenicityas compared to wild type factor FIX by the following neoantigenicitytest.

Products to be compared are administered subcutaneously into rabbitswith or in the absence of Freund's adjuvant. The endpoint assay is anative Western blot.

A suitable dose of either the FIX-HSA fusion with a linker with reducedimmunogenicity or a FIX-HSA fusion with a linker of enhancedimmunogenicity or FIX wild type can be administered as a slow bolus.

A sample can be taken about 30 to 40 days after the start of theimmunization and be assayed by a Western blot method to ensure that therabbits developed antibodies against each of the respective immunogens.

Antibodies against the test sample for which a potentially increasedimmunogenicity is to be measured are blocked by an excess of a controlsample (e.g. wild type FIX) By doing so all of the antibodies presentwhich formed against native epitopes are unable to react when thatantibody is used as a probe in a Western blot. The test and the controlsample are run on the Western blot membrane and the blocked antibody isused as a probe.

If there were epitopes on the test samples (here FIX-HSA fusion proteinwith a linker) but not in the control sample (wild type FIX) that causedantibody formation in the rabbits, these antibodies would be detectedafter blocking as residual antibodies which would react with the testsample, but not the control sample.

Likewise FIX-HSA with a linker having predicted reduced immunogenicitycan be used as a control versus a FIX-HSA with a linker having predictedincreased immunogenicity as a test sample.

Preferably the same Western blot is assayed with non-blocked antibodiesraised against the test sample as a positive control. Here it isexpected that test sample as well as control sample are detected in theWestern blot assay.

If with blocked antibodies raised against the test sample both test andcontrol sample are not detected it can be concluded that the test samplehas no neoepitopes as compared to the control sample.

If with blocked antibodies raised against the test sample only the testsample but not the control sample is detected it can be concluded thatthe test sample has neoepitopes as compared to the control sample.

For doing the analysis the IgG fraction of the hyper immune pooled serumcan be purified using a protein A column from Pierce or other supplierswith a bed volume for example greater than 1 ml according to theinstructions of the supplier. Preferably the rabbit sera are delipidatedfor example with trichlorotrifluoroethane.

Blocking is done for example by mixing 0.1-1.00 mg of the purifiedantiserum with 0.1 to 100 mg of the control sample in for example a 0.5to 5 ml centrifuge cup, bringing the volume up to 0.5 to 3 ml. The tubesare then rotated slowly at room temperature of a minimum of 2 hours.

Nonblocked control antibodies to be used as a positive control can beprepared in the same way except that no control sample is used forblocking.

All blocked and nonblocked antibodies can be added to 3 to 4.5 ml of 5%dry milk solution in TBS+0.1% Tween-20 before incubation with theirrespective membrane.

Preferably Western blots are performed as native Western blots.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: In vitro activation of FIX-albumin fusion proteins by FXIa at37° C. at a molar ratio of FXIa to fusion protein of about 1:500. Onefusion protein with non-cleavable linker (1478/797) and two fusionproteins with cleavable linker (1088/797 and 1089/797) were used.Samples were analyzed by SDS-PAGE under reducing conditions followed byCoomassie blue staining.

FIG. 2: Pharmacokinetics of activated rec FIX and FIX-albumin fusionproteins with and without cleavable linker in comparison tonon-activated fusion proteins.

FIG. 3: Inactivation of activated rec FIX or FIX-albumin fusion proteinby AT. Residual FIX activity was determined after 120 min using anon-activated partial thromboplastin time assay.

1. A fusion protein comprising: a) Factor VII (FVII), Factor VIIa(FVIIa), or Factor IX (FIX); b) a half-life enhancing polypeptide(HLEP), and c) a peptide linker which joins the FVII, FVIIa, or FIX andthe half-life enhancing polypeptide; wherein the HELP is animmunoglobulin without an antigen binding domain, wherein the peptidelinker is cleavable by one or more proteases that can activate or cleaveFVII, FVIIa, or FIX during coagulation, and wherein the fusion proteinhas at least one of the following properties, in comparison to therespective fusion protein linked by a non-cleavable linker consisting ofthe amino acid sequence GGGGGGV (SEQ ID NO: 94): i) an increased molarspecific activity in at least one coagulation-related assay, ii) anincreased inactivation rate after the peptide linker is proteolyticallycleaved in a coagulation-related mode, and iii) an increased eliminationrate after the peptide linker is proteolytically cleaved in acoagulation-related mode. 2-3. (canceled)
 4. The fusion protein of claim1, wherein said fusion protein has a higher in vivo recovery compared tothe in vivo recovery of FVII, FVIIa, or FIX when not fused to ahalf-life enhancing polypeptide.
 5. The fusion protein of claim 1,wherein said fusion protein has an increased half-life in plasmacompared to the half-life in plasma of FVII, FVIIa, or FIX when notfused to a half-life enhancing polypeptide.
 6. (canceled)
 7. The fusionprotein of claim 1, wherein the fusion protein comprises FIX. 8.(canceled)
 9. The fusion protein of claim 1, wherein the peptide linkeris cleavable by FXIa and/or FVIIa/TF.
 10. The fusion protein of claim 1,wherein the molar specific activity of the fusion protein is increasedat least 25% compared to that of the respective fusion protein linked bya non-cleavable linker consisting of the amino acid sequence GGGGGGV(SEQ ID NO: 94) in at least one coagulation-related assays.
 11. Thefusion protein of claim 1, wherein the inactivation rate of FVII, FVIIa,or FIX after cleavage of the peptide linker which links the FVII, FVIIa,or FIX to the half-life enhancing polypeptide is increased at least 10%as compared to the inactivation rate of FVII, FVIIa, or FIX in therespective fusion protein linked by a non-cleavable linker consisting ofthe amino acid sequence GGGGGGV (SEQ ID NO: 94).
 12. The fusion proteinof claim 1, wherein the elimination rate of FVII, FVIIa, or FIX aftercleavage of the peptide linker which links the FVII, FVIIa, or FIX tothe half-life enhancing polypeptide is increased by at least 10% ascompared to the elimination rate of FVII, FVIIa, or FIX in therespective fusion protein linked by a non-cleavable linker consisting ofthe amino acid sequence GGGGGGV (SEQ ID NO: 94).
 13. The fusion proteinof claim 1, wherein the peptide linker is cleavable by a proteases thatnaturally activates FVII, FVIIa, or FIX in vivo.
 14. The fusion proteinof claim 13, wherein the kinetics of the linker cleavage by the proteaseis not delayed by more than a factor of 3 compared to the kinetics ofthe activation of the FVII, FVIIa, or FIX.
 15. (canceled)
 16. The fusionprotein of claim 1, wherein the peptide linker is cleavable by FXIaand/or by FVIIa/TF, and wherein the fusion protein comprises FIX. 17.The fusion protein of claim 1, wherein the linker is cleavable by FXIaand/or by FVIIa/TF, and wherein the fusion protein comprises FVIIa. 18.The fusion protein of claim 1, wherein the linker comprises SEQ IDNO:113 or SEQ ID NO:114.
 19. A polynucleotide encoding the fusionprotein of claim
 1. 20. A plasmid or vector comprising thepolynucleotide of claim
 19. 21. The plasmid or vector of claim 20,wherein the plasmid or vector is an expression vector.
 22. The plasmidor vector of claim 20, wherein the vector is a transfer vector for usein human gene therapy.
 23. A host cell comprising a polynucleotideaccording to claim
 19. 24. A method of producing a fusion protein ofclaim 1, comprising culturing host cells comprising a polynucleotideencoding the fusion protein under conditions such that the fusionprotein is expressed.
 25. A pharmaceutical composition comprising (a)the fusion protein of claim 1, (b) a polynucleotide encoding said fusionprotein, or (c) a plasmid or vector comprising a polynucleotide encodingsaid fusion protein.
 26. A method of administering an effective amountof the fusion protein of claim 1 to a patient in need thereof,comprising (a) administering said fusion protein, (b) administering acomposition comprising a polynucleotide encoding said fusion protein viaa gene therapy protocol, or (c) administering a composition comprising aplasmid or vector comprising a polynucleotide encoding said fusionprotein via a gene therapy protocol.
 27. The method of claim 26, whereinthe patient suffers from a blood coagulation disorder.
 28. The method ofclaim 27, wherein the blood coagulation disorder is hemophilia B. 29.The method of claim 27, wherein the blood coagulation disorder is FVIIand/or FVIIa deficiency.
 30. The method of claim 27, wherein the bloodcoagulation disorder is hemophilia A.
 31. The method of claim 27,wherein the administration comprises administering via a gene therapyprotocol (a) a composition comprising a polynucleotide encoding saidfusion protein or (b) a composition comprising a plasmid or vectorcomprising a polynucleotide encoding said fusion protein.
 32. The methodaccording to claim 27, wherein the fusion protein is effective to act inthe patient as a procoagulant.
 33. The fusion protein of claim 1,wherein the FVII, FVIIa, or FIX or immunoglobulin, comprises a sequencethat is 95% identical to the sequence of a wild-type human FVII, FVIIa,or FIX or a wild-type human immunoglobulin, respectively.
 34. The methodof claim 24, further comprising recovering the fusion protein from thehost cells or from the culture medium.
 35. The method of claim 27,wherein the administration comprises administering a compositioncomprising the fusion protein of claim
 1. 36. The fusion protein ofclaim 1, wherein the linker is cleavable by FVII, FVIIa, or FIX duringcoagulation.
 37. The fusion protein of claim 1, wherein the linkercomprises a sequence from FVII, FVIIa, or FIX.
 38. The fusion protein ofclaim 1, wherein the linker is capable of being cleaved simultaneouslywith the activation of FVII, FVIIa, or FIX, or with comparable kinetics,during coagulation.
 39. The fusion protein of claim 1, wherein thefusion protein comprises FVIIa.